Information on the diet of threatened species is important in devising appropriate management plans to ensure their conservation. The Australian sea lion (Neophoca cinerea) is Australia's only endemic and globally one of the least numerous pinniped species. However, dietary information is currently limited because of the difficulty in using traditional methods (identification of prey hard parts from scats, regurgitates and stomach samples) to reliably provide dietary information. The present study assessed the use of fatty acid (FA) analysis to infer diet using milk samples collected from 11 satellite tracked Australian sea lions from Olive Island, South Australia. Satellite tracking revealed that females foraged in two distinct regions; 'inshore' regions characterised by shallow bathymetry (10.7±4.8m) and 'offshore' regions characterised by comparatively deep bathymetry (60.5±13.4m). Milk FA analysis indicated significant differences in the FA composition between females that foraged inshore compared with those that foraged offshore. The greatest differences in relative levels of individual FAs between the inshore and offshore groups were for 22:6n-3 (6.5±1.2% compared with 16.5±1.9% respectively), 20:4n-6 (6.1±0.7 compared with 2.5±0.7 respectively) and 22:4n-6 (2.4±0.2% compared with 0.8±0.2% respectively). Using discriminant scores, crustacean, cephalopod, fish and shark-dominated diets were differentiated. The discriminant scores from Australian sea lions that foraged inshore indicated a mixed fish and shark diet, whereas discriminant scores from Australian sea lions that foraged offshore indicated a fish-dominated diet, although results must be interpreted with caution due to the assumptions associated with the prey FA dataset. FA analysis in combination with satellite tracking proved to be a powerful tool for assessing broad-scale spatial dietary patterns.